PROJECT SUMMARY/ABSTRACT To date, the only vaccine trial (RV144) that has shown any protective efficacy against HIV acquisition is based on a poxvirus prime ? protein boost immunization strategy. Although the efficacy achieved was modest (~31%), these findings provide a strong rationale to seek improvements for the prime-boost immunization approach and to gain better insight on the nature of the protective immunity achieved. Correlate studies of the RV144 trial indicated that antibodies against the hypervariable loops 1 and 2 (V1/V2) region of HIV envelope protein (Env) and high levels of antibody-dependent cellular cytotoxicity (ADCC) activities inversely correlated with the risk of HIV-1 acquisition. Neutralizing antibodies (Nabs) were generated, but were primarily against sensitive (?Tier 1?) isolates, with little or no activity against the more resistant (?Tier 2?) strains typical of circulating viruses. Thus, much of the current effort in HIV vaccine research aims to elicit Tier 2 Nabs and to improve the potency and the breadth of non-neutralizing antibody responses, including V1/V2 directed antibodies and antibodies that can mediate ADCC. Using a replication-competent poxvirus for priming and gp120 for boosting, we were able to induce Nab against heterologous Tier 2 viruses as well as cross-reactive V1/V2-specific antibodies and high levels of ADCC activities in rabbits. In this application, we propose to examine novel immunogens and immunization approaches to further enhance the breadth and potency of the Nab and non-Nab responses achieved and to determine if findings in rabbits can be translated to non-human primates. We hypothesize that by presenting the Env antigen in a native trimer form with the conserved CD4 binding site unmasked, we will enhance cross-reactive Nab, and by using polyvalent Env immunogens, we will amplify responses to conserved epitopes, while broadening strain-specific responses, including those directed to variable regions. The enhanced breadth and potency of both Nab and non-Nab responses, including V1/V2- directed antibodies and those that mediate antiviral effector functions, such as ADCC, will contribute to protection against challenge in a non-human primate model. The Specific Aims are: (1) To determine if trimeric Env, instead of monomeric gp120, when used as the boosting immunogen in a prime-boost regimen may improve the breadth or potency of Nab responses; (2) To determine if the ability of a highly conserved glycan (N197) to modulate Env immunogenicity is dependent on the use of trimeric Env, instead of monomeric gp120; (3) To determine if polyvalent, rather than monovalent Env, when used in a prime-boost immunization regimen, can improve the breadth and potency of Env-specific antibody responses; and (4) To examine if immunization regimens down-selected from the preceding studies in rabbits can be translated to macaques and if the immune responses generated can protect against SHIV challenge. If successful, insights obtained from these studies will inform the clinical development of vaccines and vaccine strategies that may be more effective than those used in RV144 to prevent HIV-1 acquisition in humans.